Tick-borne bacterial agents in Hyalomma asiaticum ticks from Xinjiang Uygur Autonomous Region, Northwest China

Background Hyalomma ticks are widely distributed in semi-arid zones in Northwest China. They have been reported to harbor a large number of zoonotic pathogens. Methods In this study, a total of 334 Hyalomma asiaticum ticks infesting domestic animals were collected from four locations in Xinjiang, Northwest China, and the bacterial agents in them were investigated. Results A putative novel Borrelia species was identified in ticks from all four locations, with an overall positive rate of 6.59%. Rickettsia sibirica subsp. mongolitimonae, a human pathogen frequently reported in Europe, was detected for the second time in China. Two Ehrlichia species (Ehrlichia minasensis and Ehrlichia sp.) were identified. Furthermore, two Anaplasma species were characterized in this study: Candidatus Anaplasma camelii and Anaplasma sp. closely related to Candidatus Anaplasma boleense. It is the first report of Candidatus Anaplasma camelii in China. Conclusions Six bacterial agents were reported in this study, many of which are possible or validated pathogens for humans and animals. The presence of these bacterial agents may suggest a potential risk for One Health in this area. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06256-y.


Background
In past decades, tick and tick-borne diseases have posed a great threat to livestock productivity and public health globally.To date, 20 tick genera belonging to three families (Ixodidae, Argasidae, and Nuttalliellidae) have been defined worldwide [1,2].Of those, the ixodid tick species of the genus Hyalomma is mainly distributed in the semi-arid zones (such as semidesert steppes, savannas, scrubland, etc.) of African, Asian, and Mediterranean countries [3].A total of 27 Hyalomma species are recognized, presenting on all continents except in North and South America [3].Most Hyalomma ticks are three-host ticks; in each of their three stages, they must find a new host to take the blood meal [4].The hosts of Hyalomma ticks are mainly domestic or wild animals.Occasionally, humans can also become their accidental hosts.
Hyalomma ticks (Hyalomma anatolicum, Hy. marginatum, Hy. rufipes, Hy. asiaticum, etc.) have been recognized as the competent vector of Crimean Congo hemorrhagic fever virus (CCHFV), which causes an acute and often fatal hemorrhagic fever in humans, with the reported fatality rate ranging from 3 to 30% [5].Apart from CCHFV, many viruses, bacteria, and protozoan agents have been reported in Hyalomma ticks, such as West Nile virus, Rift Valley fever virus, Rickettsia rickettsii, R. aeschlimannii, R. sibirica, Anaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii, Borrelia turcica, Babesia occultans, and Theileria ovis [4,5], many of which cause zoonotic diseases.However, for many of these agents, the roles of Hyalomma ticks in their transmission and maintenance are still to be determined.
In China, the major species in the genus Hyalomma are Hyalomma scupense, Hy. asiaticum, Hy. dromedarii, Hy. anatolicum, and Hy.marginatum [6].They are mainly distributed in northwestern provinces such as Xinjiang, Gansu, and Inner Mongolia.Although many investigations studying tick-borne agents in Hyalomma ticks have been carried out in China, comprehensive studies on bacterial agents are still limited given their vast geographical distribution.In Xinjiang Uygur Autonomous Region, with an area of 1,664,900 km 2 , although some studies reported the agents in Hyalomma ticks, most are still preliminary and lack further genetic characterization.In this study, we collected ticks in four locations in Xinjiang and investigated the tick-borne bacterial agents in them.

Sample collection and DNA extraction
From April to May 2023, ticks were collected from the body surface of domestic animals in four locations in Xinjiang Uygur Autonomous Region (Qitai, Mulei, and Hutubi counties of Changji Hui Autonomous Prefecture and 150 Regiment of Shihezi City) (Fig. 1).Ticks were carefully removed from domestic animals (camels, sheep, and cattle) using tweezers.All ticks were brought to the laboratory alive.Morphological identification was performed to determine their species initially [7].After washing twice using phosphate buffer, all the ticks were ground into homogenate and subjected to DNA extraction using the TIANamp genomic DNA Extraction Kit (TIANGEN Co.).The tick species was confirmed by amplifying and sequencing the mitochondrial cytochrome C oxidase subunit I (COI) gene (primers shown in [8]).

Detection of Borrelia and analysis
All the DNA samples were screened for Borrelia using primers amplifying the flaB (flagellin B) gene, with a PCR product of approximately 400 bp.For the positive samples, partial 16S rRNA (approximately 1200 bp) sequences were amplified for further characterization (primers shown in [9]).All obtained sequences were aligned with those in the GenBank Database by BLASTn to determine the nucleotide identity.For phylogenetic analysis, reference sequences were downloaded from the GenBank Database and manually aligned with those recovered in this study using the MegAlign program.The Fig. 1 Map showing the four locations where the Hyalomma asiaticum ticks were collected in Xinjiang maximum likelihood (ML) tree was then constructed using PhyML v3.0 under the best-fit substitution model determined by MEGA 7.0 [10].

Detection and analysis of Rickettsia and Anaplasmataceae bacteria
Rickettsia and Anaplasmataceae bacteria (mainly Anaplasma and Ehrlichia) were detected by hemi-nested or nested PCR targeting a partial region of the 16S rRNA gene (approximately 900 bp for Rickettsia and 500 bp for Anaplasmataceae bacteria) [11].After sequencing and alignment by BLASTn, the bacterial species were initially determined.For further characterization, the gltA (citrate synthase) and groEL (60 kDa chaperonin) sequences were recovered from samples positive for Rickettsia, Ehrlichia, and Anaplasma using degenerate primers (primers shown in [11]) or specific primers (primers shown in [12]).The ompA sequences were additionally obtained from Rickettsia strains, while longer 16S sequences (approximately 800 bp) were obtained from Anaplasma strains [11].All the sequences were genetically and phylogenetically analyzed as indicated above.

Samples
In the summer of 2023, 334 ticks were collected in four locations in Xinjiang.In Mulei County, 72 ticks were collected from camels.In Hutubi County and Shihezi City, 46 and 96 ticks were collected from goats, respectively.In Qitai County, 120 ticks were collected from cattle.All ticks were initially identified to be Hy.asiaticum based on their morphology by using taxonomic keys.This result was confirmed by sequencing and analysis of the COI sequences (all sequences showed > 99% identities to Hy. asiaticum).

Rickettsia sp.
Three ticks tested positive for Rickettsia, one from Qitai County and two from Mulei County.The 16S sequences are 100% identical to R. sibirica strain 246 and R. sibirica subsp.mongolitimonae HA-91.The gltA, groEL, and ompA sequences were also analyzed, which are closely related to R. sibirica subsp.mongolitimonae strains (Fig. 3) with 100%, 99.90%, and 100% nucleotide similarities.Therefore, these strains are determined to be R. sibirica subsp.mongolitimonae.

Ehrlichia spp.
A total of 12 Ehrlichia strains were detected in the 334 ticks (3.59%), representing two species.Of those, four strains (QT54, QT88, QT98, QT99) from Qitai County were determined to be E. minasensis, with their 16S, gltA, and groEL sequences showing 100%, 99.40-99.60%,and 100% identities to E. minasensis strains.Analysis of the other eight Ehrlichia strains indicated that they are closely related to Ehrlichia sp.BL116-8, which was also identified in ticks from Xinjiang.Their 16S and gltA sequences are all 100% identical to Ehrlichia sp.BL116-8.Interestingly, as shown in the phylogenetic tree, although their 16S and gltA are distinct from E. minasensis, all their groEL sequences are closely related to those of E. minasensis strains (99.81-100% nucleotide identity) (Fig. 4).

Anaplasma spp.
Based on the 16S, gltA, and groEL sequences, two Anaplasma species were initially determined: One is almost identical to Anaplasma sp.BL099-6 previously reported in Bole City of Xinjiang.Meanwhile, it is also closely related to Candidatus Anaplasma boleense in the phylogenetic trees.In our opinion, it might be reasonable to classify it as a subspecies or variant of Candidatus Anaplasma boleense.For the other species, only 16S and groEL sequences were successfully obtained.The 16S sequences are highly homologous to Candidatus Anaplasma camelii and Candidatus Anaplasma cinensis (both have 99.87-100% identity), while the groEL sequences have highest 95.44% nucleotide similarity to Anaplasma sp.CL BL 90 and 94.90% to Candidatus Anaplasma camelii strains.In the phylogenetic trees, they are also closely related to Candidatus Anaplasma camelii strains (Fig. 5).Based on these data, we propose that they should be a variant of Candidatus Anaplasma camelii despite the absence of gltA sequences.

Discussion
Hyalomma ticks are important vectors of human pathogens in Northwest China.However, extensive and comprehensive studies on the bacterial pathogens they harbor are still lacking.In this study, we collected 334 Hy. asiaticum ticks from four locations in Xinjiang, and six bacterial agents were detected and identified.
Since 2014, the former genus Borrelia has been divided into two genera: the genus Borrelia containing the members of the relapsing fever Borrelia and the genus Borreliella containing the Lyme disease Borreliella [13].In China, Borreliella burgdorferi sensu lato, the agent of Lyme disease, has been frequently detected in multiple tick species [6,14].However, reports on members of the current genus Borrelia have been quite few in Xinjiang.In 2020, Zhao et al. reported a previously undescribed Borrelia species in Rhipicephalus turanicus from Xinjiang based on the groEL sequence [15].In 2022, a nationwide survey revealed the remarkable genetic diversity of relapsing fever Borrelia in ticks from China [16].However, no Hy.asiaticum has been reported to harbor relapsing fever Borrelia.In this study, we detected an undescribed Borrelia species in Hy. asiaticum ticks from all four locations, suggesting its wide distribution in Xinjiang.Analysis of its 16S and flaB genes indicated that it is genetically distant from known Borrelia species.Its pathogenicity to humans is still unclear and warrants further investigation.
The only Rickettsia species identified in this study is R. sibirica subsp.mongolitimonae, a member of the spotted group Rickettsia.Rickettsia sibirica subsp.mongolitimonae has long been recognized as a human pathogen causing fever, lymphangitis, septic shock, encephalitis, and retinal vasculitis [17][18][19].Although it was first isolated from Hy. asiaticum ticks in Inner Mongolia of China [20], it was mostly reported in European and African countries, such as France, Spain, Portugal, Greece, Algeria, South Africa, and Cameroon [17-19, 21, 22].As previously reported, R. sibirica has been reported in Xinjiang as well as elsewhere in China; however, almost all of them are R. sibirica subsp.sibirica or only identified to species level [23][24][25][26].So far as we know, this may be the second report of R. sibirica subsp.mongolitimonae in China.
Two Ehrlichia species were identified in this study.Intriguingly, the two Ehrlichia species share almost the same groEL gene sequences (99.81-100%) despite the obvious discrepancy in their 16S and gltA sequences.We suppose that this may result from frequent recombination between different Ehrlichia species, which may be the driving force of their adaptivity and virulence.Besides, so far as we know, this is the first description of E. minasensis in Xijiang.Ehrlichia minasensis is a tick-borne agent infecting cattle, cervids, and dogs [27].Infection with E. minasensis in calf results in clinical manifestations such as fever, depression, lethargy, and thrombocytopenia [28].It should be noted that Xinjiang is one of the most important provinces of animal husbandry in China, and the presence of E. minasensis may pose a great threat to animal health.Surveillance in domestic animals and vector control measures should be carried out to prevent its transmission.Furthermore, although the two Ehrlichia species have not been reported to infect humans, whether the Candidatus Anaplasma camelii was first described in one-humped camels from Morocco in 2017 [29].Since then, it has been detected in camels and their associated ectoparasites (camel keds and ticks) from Kenya and Saudi Arabia [30,31].In 2018, it was also detected in cattle from Peninsular Malaysia [32], suggesting its wide geographic distribution and wide host range.In this study, it is of interest that all Candidatus Anaplasma camelii strains were detected in Hy. anatolicum ticks infesting apparently healthy cattle from Qitai County.There may be two possibilities: (1) The Hy. anatolicum ticks may be vectors of Candidatus Anaplasma camelii.Previously, only one study reported the presence of Candidatus Anaplasma camelii in ticks.It has been demonstrated that camel keds (Hippobosca camelina) act as vectors of this Anaplasma species [33].Instead, the role of Hy. anatolicum ticks in the transmission of Candidatus Anaplasma camelii has never been proved.(2) The detected DNA of Candidatus Anaplasma camelii is from the blood of cattle infected with it.Namely, cattle may be the reservoir of Candidatus Anaplasma camelii.This is consistent with a previous study in Peninsular Malaysia [32].Both possibilities are worth further consideration and investigation.This is also the first report of Candidatus Anaplasma camelii in China.The presence of two Anaplasma species in this study should raise concern in Xinjiang due to their veterinary importance.
There are also some limitations in the current study.All the agents were detected in ticks collected from domestic animals.The possibility cannot be ruled out that the agents were actually from the blood meal of ticks.In other words, it is still not clear whether the Hy.asiaticum ticks are the effective vector of these bacteria.In future studies, investigations of these agents in host-seeking ticks at different developmental stages and domestic animals are still needed.

Conclusions
Our results indicated the distribution and diversity of tick-borne agents in Hy. anatolicum ticks from Xinjiang, Northwest China.Among the six bacterial agents identified in this study, one represents a putative novel species (Candidatus Borrelia hyalommii), one is a human pathogen (Rickettsia sibirica subsp.mongolitimonae), and some of the others are animal pathogens.Possible recombination has been observed between Ehrlichia species,

Fig.
Fig. Phylogenetic trees based on the flaB and 16S sequences of Candidatus Borrelia hyalommii strains

Fig. 5
Fig. 5 Phylogenetic trees based on the 16S, gltA, and groEL gene sequences of the Anaplasma strains

Table 1
Prevalence of tick-borne bacterial agents in Hyalomma asiaticum in four locations of Xinjiang Uygur Autonomous Region a Positive samples/total samples Phylogenetic trees based on the 16S, gltA, and groEL gene sequences of the Ehrlichia strains which may contribute to their pathogenicity.Furthermore, we also reported the presence of Candidatus Anaplasma camelii in China for the first time.All these data suggested the potential risk for human and animal health in this area.